The melanocortin 1 receptor (MC1R) and p16 INK4A (p16) are bona fide melanoma predisposition genes, with mutations in p16 having the highest impact on risk, and MC1R loss of function (LOF) variants being the most common in melanoma patients and melanoma prone individuals. The genetic impact of co-inheritance of p16 and MC1R mutations is supported by compelling epidemiological evidence, but the molecular mechanisms by which the interaction of these two genes impacts melanoma risk is not well understood. Earlier work has attributed their combined effects to cell cycle deregulation and DNA repair deficiencies. Our team has discovered that these two risk factors share molecular mechanisms that also involve the antioxidant capacity of melanocytes. Our extensive experience in investigating the functions of MC1R and p16 puts us in the best position as a team to further characterize the mechanisms by which inheritance of mutations in both genes synergistically contributes to melanoma risk. Using unique primary cultures of skin cells including melanocytes derived from patients with mutations in both p16 and MC1R provides us with the unprecedented opportunity to identify novel common pathways that can be targeted for melanoma prevention and treatment. We propose the hypothesis that co-inheritance of germline mutations in p16 and MC1R causes synergistic disruption of the antioxidant and DNA repair responses of melanocytes to UV, and deficiencies in these protective mechanisms underlie the extremely high risk for melanoma observed in individuals who carry mutations in both genes. This hypothesis will be tested in two Specific Aims, 1) to evaluate the effects of p16 mutations and/or MC1R RHC variants on p16 expression and function in regulation of the cell cycle and senescence, repair of DNA damage, and oxidative stress, using cultured melanocytes and 3-D skin construct, and 2) to determine the effects of p16 and/or MC1R mutations on gene expression in UV-irradiated primary melanocyte cultures using RNA sequencing. The prevention and treatment opportunities identified will have significant impact not only in the high-risk patients who have donated tissue for the work proposed here, but on all persons at risk for this deadly disease.